Abstract:

A high-throughput screening method based on membrane for selecting cholesterol oxidase mutants with improved thermal activity was proposed. This method takes advantage of the fact that the process of enzyme treatment and the reaction between enzyme and substrate was separated effectively，thus preventing the enzyme from reaction with substrate before heat treatment. High temperature treatment only involved the membrane not the agar plate，thereby the problem of softening or melting of the agar plate was resolved. This method is applicable to the screening of mutant colonies with high activity which produce obvious reddish brown transparent circle on the agar plate. The nitrocellulose filter membrane used for transfer printing is cheap and the major advantages of this method are reliability and simple，rapid screening.